本文關(guān)鍵詞： 創(chuàng)傷性腦損傷 亞甲藍(lán) 神經(jīng)元 腦水腫 神經(jīng)功能缺損 創(chuàng)傷性腦損傷 亞甲藍(lán) 促炎細(xì)胞因子 小膠質(zhì)細(xì)胞 自噬 mTOR/p70S6K　出處：《浙江大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：研究背景： 創(chuàng)傷性腦損傷是一種嚴(yán)重危及人類健康的疾病,有著較高的發(fā)病率、致殘率和死亡率。全世界每年約有1000多萬(wàn)人遭受各種類型和程度的顱腦損傷。創(chuàng)傷性顱腦損傷不僅導(dǎo)致患者死亡,即使有幸存活的患者也會(huì)在傷后遭受不同程度的并發(fā)癥,包括各種神經(jīng)功能缺損和運(yùn)動(dòng)功能的障礙等。 最近數(shù)十年來(lái),隨著急診搶救與重癥監(jiān)護(hù)設(shè)備、技術(shù)以及藥物的進(jìn)展,腦損傷患者的生存率得到了較大的改善。不過(guò)令人遺憾的是,到目前為止,仍然沒(méi)有一種藥物可以很好的防止顱腦損傷導(dǎo)致的各種長(zhǎng)遠(yuǎn)期的并發(fā)癥。一系列的研究已經(jīng)表明,顱腦損傷的損害的嚴(yán)重程度不僅是和原發(fā)性創(chuàng)傷相關(guān)的,更多的是和繼發(fā)性損傷密切相關(guān),包括神經(jīng)炎癥反應(yīng)、腦水腫和血腦屏障的破壞等等。繼發(fā)性損傷可以從損傷開(kāi)始后不久一直持續(xù)幾天、幾個(gè)月甚至幾年,最終導(dǎo)致神經(jīng)元的死亡或神經(jīng)退行性疾病的發(fā)生。另一方面,原發(fā)性顱腦損傷在損傷當(dāng)時(shí)就已經(jīng)發(fā)生,在臨床上是無(wú)法干預(yù)的。因此,目前腦損傷的研究都集中在如何減少繼發(fā)性顱腦損傷。尋找合適的藥物靶點(diǎn)及藥物干預(yù)繼發(fā)性顱腦損傷,是改善顱腦損傷患者預(yù)后的關(guān)鍵。 亞甲藍(lán)是一種噻吩嗪類化合物,具有自身氧化還原的特性。其在臨床已經(jīng)使用了120多年,既往主要用于治療高鐵血紅蛋白血癥、瘧疾、氰化物中毒、心肺轉(zhuǎn)流術(shù)和膿毒血癥。近年來(lái),亞甲藍(lán)在中樞神經(jīng)系統(tǒng)中的作用越來(lái)越引起大家的重視,亞甲藍(lán)對(duì)于缺血性腦卒中、側(cè)索性硬化、阿爾茨海默氏病、帕金森病、Leber視神經(jīng)病變和其他神經(jīng)退行性疾病等都具有潛在的治療價(jià)值。由于顱腦損傷和其他類型的神經(jīng)系統(tǒng)病變?nèi)缛毖阅X卒中和退行性神經(jīng)病變方面有不少共同的病理生理機(jī)制,因此,我們推測(cè)亞甲藍(lán)在顱腦損傷中也具有同樣的治療價(jià)值。 本研究擬采用ICR小鼠進(jìn)行創(chuàng)傷性顱腦損傷的造模,探討亞甲藍(lán)對(duì)創(chuàng)傷性顱腦損傷的神經(jīng)保護(hù)作用并進(jìn)一步研究其可能的作用機(jī)制。 第一部分亞甲藍(lán)對(duì)創(chuàng)傷性顱腦損傷的神經(jīng)保護(hù)作用 研究目的： 本研究采用ICR小鼠進(jìn)行創(chuàng)傷性腦損傷造模,探討亞甲藍(lán)是否對(duì)創(chuàng)傷性顱腦損傷具有神經(jīng)保護(hù)的作用。 研究方法： (1)預(yù)實(shí)驗(yàn)確定亞甲藍(lán)的最佳使用劑量：將小鼠分為5組,分別為假手術(shù)組、生理鹽水處理組、低劑量亞甲藍(lán)治療組(0.lmg/kg)、中等劑量亞甲藍(lán)治療組(l.0mg/kg)和高劑量亞甲藍(lán)治療組(10mg/kg)。以腦損傷后24小時(shí)腦含水量和神經(jīng)功能缺損評(píng)分為依據(jù),確定亞甲藍(lán)的最佳劑量。 (2)根據(jù)預(yù)實(shí)驗(yàn)結(jié)果,將小鼠分為假手術(shù)組、生理鹽水處理組和亞甲藍(lán)治療組。結(jié)合文獻(xiàn)資料,我們選擇傷后24小時(shí)及72小時(shí)進(jìn)行神經(jīng)功能缺損評(píng)分、腦損傷體積、腦含水量和神經(jīng)元數(shù)量的檢測(cè)；在傷后14天進(jìn)行神經(jīng)功能缺損評(píng)分和腦損傷體積的測(cè)定。 (3)相關(guān)指標(biāo)評(píng)估的方法：采用改良的神經(jīng)功能缺損評(píng)分進(jìn)行神經(jīng)功能評(píng)估；采用干濕重量法測(cè)定腦含水量；采用免疫組化NeuN染色,用Image-Pro Plus軟件檢測(cè)神經(jīng)元的數(shù)量；采用連續(xù)切片HE染色,用Image tool軟件計(jì)算腦損傷體積。 研究結(jié)果： (1)預(yù)實(shí)驗(yàn)結(jié)果表明：腦損傷后損傷側(cè)大腦半球腦含水量及神經(jīng)功能缺損較假手術(shù)組明顯增高(P0.05)。與生理鹽水處理組比較,采用l.Omg/kg亞甲藍(lán)治療后能顯著降低傷后24小時(shí)的神經(jīng)功能缺損評(píng)分和腦含水量(P0.05),亞甲藍(lán)其他兩個(gè)劑量與生理鹽水組比較都無(wú)統(tǒng)計(jì)學(xué)差異(P0.05)。因此確定使用亞甲藍(lán)1.0mg/kg為我們正式實(shí)驗(yàn)的劑量。 (2)腦含水量的檢測(cè)結(jié)果表明,亞甲藍(lán)可以顯著降低ICR小鼠創(chuàng)傷性腦損傷后24小時(shí)損傷側(cè)大腦半球的腦含水量(P0.05),而對(duì)側(cè)大腦半球、腦干、小腦等部位的腦含水量無(wú)統(tǒng)計(jì)學(xué)差異(P0.05)。同時(shí)我們發(fā)現(xiàn)在傷后72小時(shí)兩組小鼠在各個(gè)部位的腦含水量無(wú)統(tǒng)計(jì)學(xué)差異(P0.05)。 (3)神經(jīng)元的免疫組化檢測(cè)表明,采用亞甲藍(lán)治療的小鼠在傷后24小時(shí)及72小時(shí)損傷區(qū)的神經(jīng)元數(shù)量要顯著高于生理鹽水處理組,兩者存在著統(tǒng)計(jì)學(xué)的差異(P0.05)。 (4)神經(jīng)功能缺損評(píng)分表明,與生理鹽水處理組相比,采用亞甲藍(lán)治療能降低小鼠腦損傷后24小時(shí)和72小時(shí)的神經(jīng)功能缺損評(píng)分,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。而在腦損傷后14天,兩組小鼠的神經(jīng)功能缺失評(píng)分無(wú)顯著差異(P0.05)。 (5)腦損傷體積檢測(cè)結(jié)果表明,與生理鹽水組比較,亞甲藍(lán)治療減少了腦損傷后24小時(shí)、72小時(shí)和14天腦損傷的體積(P0.05)。 結(jié)論： 亞甲藍(lán)治療可以降低創(chuàng)傷性腦損傷的神經(jīng)功能缺損評(píng)分,降低創(chuàng)傷性腦損傷引起的腦水腫,減少腦損傷的體積,阻止創(chuàng)傷性腦損傷后神經(jīng)元的死亡。第二部分亞甲藍(lán)對(duì)創(chuàng)傷性顱腦損傷的神經(jīng)保護(hù)作用機(jī)制探討 研究目的：探討亞甲藍(lán)對(duì)創(chuàng)傷性顱腦損傷神經(jīng)保護(hù)作用的可能機(jī)制。 研究方法： (1)我們將實(shí)驗(yàn)小鼠分為假手術(shù)組、生理鹽水處理組和亞甲藍(lán)治療組。結(jié)合文獻(xiàn)資料,在傷后24小時(shí)及72小時(shí)評(píng)估自噬激活情況、促炎細(xì)胞因子水平和小膠質(zhì)細(xì)胞激活情況；在傷后14天評(píng)估小膠質(zhì)細(xì)胞激活情況。 (2)相關(guān)指標(biāo)評(píng)估的方法：免疫組化Beclin-1染色,Western Blot檢測(cè)Beclin-1和LC3Ⅱ/Ⅰ來(lái)評(píng)估自噬激活情況；Western Blot來(lái)檢測(cè)炎癥因子IL-6、 ILlβ的表達(dá)量； Iba-1免疫組化染色來(lái)檢測(cè)小膠質(zhì)細(xì)胞激活情況。 (3) mTOR/p70S6K通路中相關(guān)蛋白的變化情況采用Western Blot檢測(cè)。 研究結(jié)果： (1)腦損傷后自噬顯著激活；與生理鹽水處理組比較,亞甲藍(lán)治療促進(jìn)了創(chuàng)傷性腦損傷后24小時(shí)及72小時(shí)自噬的發(fā)生,表現(xiàn)為Beclin-1表達(dá)的上調(diào)和LC3Ⅱ/Ⅰ比值的增加(P0.05)。 (2)腦損傷后促炎細(xì)胞因子IL-6、IL1β的釋放增加；與生理鹽水處理組比較,亞甲藍(lán)顯著抑制了腦損傷后24小時(shí)及72小時(shí)IL-6、IL1β的表達(dá)(P0.05)。 (3)小膠質(zhì)細(xì)胞在腦損傷后72小時(shí)和14天激活明顯(P0.05)；與生理鹽水處理組相比,亞甲藍(lán)治療抑制了創(chuàng)傷性腦損傷后72小時(shí)及14天小膠質(zhì)細(xì)胞的激活(P0.05)。 (4)腦損傷后1nTOR/p70S6K通路激活,表現(xiàn)為磷酸化的mTOR和磷酸化的p70S6K表達(dá)增加(P0.05)。而與生理鹽水處理組相比,亞甲藍(lán)治療后顯著抑制了mTOR/p70S6K通路中磷酸化的mTOR及磷酸化的p70S6K蛋白的表達(dá)(P0.05),而對(duì)于總的mTOR及總的p70S6K蛋白的水平無(wú)明顯的影響(P0.05)。 結(jié)論：(1)亞甲藍(lán)治療可以通過(guò)減少創(chuàng)傷性腦損傷后促炎細(xì)胞因子的釋放,抑制小膠質(zhì)細(xì)胞的激活及促進(jìn)自噬的發(fā)生起到神經(jīng)保護(hù)作用。(2)結(jié)合文獻(xiàn)資料和以上實(shí)驗(yàn)結(jié)果,我們認(rèn)為亞甲藍(lán)可能是通過(guò)抑制mTOR/p70S6K通路相關(guān)蛋白的激活,從而起到了抑制神經(jīng)炎癥反應(yīng)和促進(jìn)自噬的作用。
[Abstract]:Research background:
Traumatic brain injury is a serious threat to human health disease, with high morbidity, disability and mortality. There are about about 10000000 people suffering from traumatic brain injury and the degree of various types in the world each year. Not only leads to death in patients with traumatic brain injury, even survive patients will suffer different degrees of complications in trauma after including the deficit and motor function in various neurological disorders.
In recent decades, with the emergency and intensive care equipment, technology and drug development, the survival rate of patients with brain injury has been greatly improved. But unfortunately, so far, there is not a drug can prevent brain injury caused by long-term complications as well. A series of research have shown that the severity of craniocerebral injury and damage is not only the primary injury related, is more closely related and secondary injury, including nerve inflammation, brain edema and blood brain barrier damage. Following the injury from injury shortly after continued for several days, or even months in recent years, resulting in the death of neurons or neurodegenerative diseases. On the other hand, primary craniocerebral injury in the injury had occurred, in the clinic is not intervention. Because of this, the brain The research of injury is focused on how to reduce secondary craniocerebral injury. Finding the right drug targets and drugs to intervene secondary craniocerebral injury is the key to improve the prognosis of patients with craniocerebral injury.
Methylene blue is a phenothiazine compounds, has the characteristics of self oxidation reduction. It has been used for 120 years in the clinical history, mainly used for the treatment of methemoglobinemia, malaria, cyanide poisoning, cardiopulmonary bypass and sepsis. In recent years, methylene blue and function in the central nervous system more attention, methylene blue for ischemic stroke, side simply sclerosis, Alzheimer's disease, Parkinson's disease, has the potential therapeutic value of Leber optic neuropathy and other neurodegenerative diseases. Due to disease of the central nervous system and other types of brain injury such as ischemic stroke and neurodegenerative diseases are many common pathophysiological mechanisms, therefore, we speculate that methylene blue has the same therapeutic value in traumatic brain injury.
The purpose of this study is to establish a model of traumatic brain injury in ICR mice, and to explore the neuroprotective effect of methylene blue on traumatic brain injury and to further study its possible mechanism.
The neuroprotective effect of methylene blue on traumatic brain injury in the first part
The purpose of the study is:
In this study, ICR mice were used to make a model of traumatic brain injury and to explore the effect of methylene blue on the neuroprotective effect of traumatic brain injury.
Research methods:
(1) methylene blue dosage determined pre experiment: mice were divided into 5 groups, namely sham operated group, normal saline group, low dose of methylene blue treatment group (0.lmg/kg), medium dose of methylene blue (l.0mg/kg) treatment group and high-dose treatment group of methylene blue (10mg/kg) based on the water and nerve function. 24 hours after brain injury with cerebral impairment score as the basis, to determine the optimal dose of methylene blue.
(2) according to the results of the experiment, the mice were divided into sham operation group, saline treatment group and methylene blue group. Combined with the literature, we selected 24 hours after injury and 72 hours of neurological deficit score, brain injury volume, detection of brain water content and the number of neurons in the neural function defect determination; score and brain injury Volume 14 days after injury.
(3) method evaluation: the neural function defect score on the modified neurological function assessment; brain water content was determined by dry wet weight method; by using immunohistochemical NeuN staining, with the number of Image-Pro Plus software to detect neurons; using serial sections of HE staining, calculate the brain injury volume using Image tool software.
The results of the study:
(1) the results of pre experiments show that: after brain injury, ipsilateral hemisphere brain water content and neurological function were significantly higher than those in the sham operation group (P0.05). With the saline treated group compared with l.Omg/kg methylene blue after treatment can significantly reduce the injury of nerve function defect score and 24 hours of brain water content (P0.05), methylene blue, the other two dose compared with normal saline group had no significant difference (P0.05). It was determined using methylene blue 1.0mg/kg as our formal experimental dose.
(2) the detection results of cerebral water content showed that methylene blue can significantly reduce the amount of water for 24 hours the ipsilateral cerebral hemisphere of brain after traumatic brain injury in mice with ICR (P0.05), and the contralateral cerebral hemisphere, brainstem, cerebellum and other parts of the brain water content had no significant difference (P0.05). At the same time we found in in various parts of the brain injury after 72 hours in two groups were no significant difference in water content (P0.05).
(3) immunohistochemistry showed that the number of neurons in the injured area treated by methylene blue was significantly higher than that in saline group at 24 hours and 72 hours after injury, and there was a significant difference between them (P0.05).
(4) the neurological deficit scores showed that compared with normal saline group, using methylene blue treatment can reduce brain injury in mice after nerve function defect in 24 hours and 72 hours of score, the difference was statistically significant (P0.05). The brain injury after 14 days, there was no significant difference between the scores of neurological deficits in two groups of mice the (P0.05).
(5) the results of brain damage volume test showed that methylene blue therapy reduced the volume of brain damage (P0.05) at 24 hours, 72 hours and 14 days after brain damage compared with the saline group.
Conclusion:
Methylene blue treatment can reduce neurological function after traumatic brain injury caused by traumatic brain injury, decrease brain edema and reduce the volume of brain damage, prevent traumatic neuronal death after brain injury. The second part of the research of methylene blue on traumatic brain injury of the nerve protection mechanism
Objective: To investigate the possible mechanism of methylene blue on the neuroprotective effect of traumatic brain injury.
Research methods:
We (1) mice were randomly divided into sham operation group, saline treatment group and methylene blue group. According to the data, in 24 hours and 72 hours to assess the activation of autophagy after injury, levels of proinflammatory cytokines and activation of microglia activation; on the 14 day after injury assessment of microglia.
(2) related indicators evaluation methods: immunohistochemistry Beclin-1 staining, Western Blot detection Beclin-1 and LC3 II / I to evaluate autophagy activation; Western Blot to detect inflammatory factor IL-6, ILl beta expression; Iba-1 immunohistochemical staining to detect microglia activation.
(3) the changes of related proteins in the mTOR/p70S6K pathway were detected by Western Blot.
The results of the study:
(1) autophagy was significantly activated after brain injury. Compared with saline group, methylene blue treatment promoted the occurrence of autophagy at 24 hours and 72 hours after traumatic brain injury, which was manifested by the up regulation of Beclin-1 expression and the increase of LC3 II / I ratio (P0.05).
(2) the release of proinflammatory cytokines IL-6 and IL1 beta increased after brain injury. Compared with saline group, methylene blue significantly inhibited the expression of IL-6 and IL1 IL1 at 24 hours and 72 hours after brain injury.
(3) microglia activated significantly in 72 hours and 14 days after brain injury (P0.05). Compared with saline group, methylene blue treatment inhibited microglial activation (P0.05) at 72 hours and 14 days after traumatic brain injury.
(4) the activation of 1nTOR/p70S6K after brain injury, manifested as mTOR phosphorylation and phosphorylation of the increased expression of p70S6K (P0.05). Compared with the normal saline group, methylene blue treatment significantly inhibited the phosphorylation of mTOR and phosphorylation of mTOR/p70S6K pathway of p70S6K protein expression (P0.05), and for the total mTOR and total p70S6K protein levels had no significant effect (P0.05).
Conclusion: (1) methylene blue treatment can reduce traumatic brain injury after the release of proinflammatory cytokines and inhibit the activation of microglia and promote autophagy plays a neuroprotective role. (2) combined with literature data and experimental results, we believe that the methylene blue probably by inhibiting activation of mTOR/p70S6K pathway protein, so as to inhibit nerve inflammation and promote autophagy.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R651.15

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本文編號(hào)：1500824